Folie 1

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Valproic acid in SMA
Brunhilde Wirth Institute of Human
Genetics University of Cologne, Germany
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From Gene to protein
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SMN genes, transcripts and proteins
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Alternative splicing of SMN2 RNA
SMN1
ttattttccttacagGGTTTCAGACAAAATCAAAAAGAAGGAAGG
AGgt
E8
E6
GGTTTTAGACAAAATCAAAAAGAAGGAAGG
SMN2
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Correct splicing of SMN2 is restored by
overexpression of Htra2-ß1 and Hra2-ß1
interacting splicing factors
Hofmann et al., PNAS 2000 Hofmann and Wirth Hum
Mol Genet 2002
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How to increase the SMN2 protein expression ?
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Therapeutical strategies for SMA
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HDAC inhibitors

• 1. Short chain fatty acids
• Valproic acid (VPA)
• Phenylbutyrate (BP)
• Sodium butyrate (NaBu)
• 2. Hydroxamic acids
• TSA
• SAHA
• 3. Benzamides
• MS275
• M344

VPA
SAHA
M344
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HDAC inhibitors activate gene transcription
transcription
no methylation acetylation
methylation no acetylation
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Rational for valproic acid as a potential
therapy for SMA

• Treatment of SMA with butyrate
• Chang et al. PNAS (2001)

short chain fatty acid, HDAC inhibitor BUT very
short half life in vivo (6 min in serum)

• Valproic acid (VPA)
• another short chain fatty acid, HDAC inhibitor
• Phiel et al. J Biol Chem (2001), Göttlicher et
  al. Embo J (2001)

FDA approved drug, successfully used in epilepsy
treatment for decades, half life 8 to 10 h in
human serum
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VPA increases SMN protein levels depending on the
SMN2 copy number
SMN maximum
2.7x
3.1x
4.3x
Brichta et al., Hum. Mol. Genet. 2003,19,
2481-2489
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VPA mediates reversion of ?7-SMN2 RNA into
FL-SMN2 RNA via Htra2-ß1
Htra2-ß1 maximum
2.7x
2.7x
4.1x
Brichta et al., Hum. Mol. Genet. 2003,19,
2481-2489
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Mechanism of action of valproic acid on the SMN2
gene

• The SMN2 transcription level is increased by
  promotor activation most likely through AP1 and
  Sp1 elements
• ?7-SMN2 RNA is restored into FL-SMN2 RNA most
  likely due to increased levels of Htra2-ß1

Brichta et al., Hum. Mol. Genet. 2003,19,
2481-2489
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Drug validation
RAT hippocampal brain slices
HUMAN hippocampal brain slicesderived from
epilepsy surgery
ß-actin (42 kDa)
ß-actin (42 kDa)
rat smn (40 kDa)
human SMN (40 kDa)
control
VPA
control
VPA
Brichta et al., Hum Mol Genet. 2003 Hahnen
et al., submitted
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SAHA increases SMN protein level in SMA
fibroblasts
SAHA
- Transcription activation - Very modest
correction of splicing
Hahnen et al., submitted
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Drug validation
Human brain slices derived from epilepsy surgery
P1
P2
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Treatment of SMA fibroblasts with M344
M344
Hahnen et al., submitted
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Treatment of rat cultured motor neurons with
VPA, M344 and SAHA
Hahnen et al., submitted
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Clinical trial with VPA in SMA parents

• 10 parents of type I-III SMA patients
• All have one SMN1 and 1-3 SMN2 copies.
• Treatment for about 4-5 months (70-80 µg VPA/ml
  serum)
• 9 measurements of SMN2-RNA and SMN-protein from
  blood samples
• Results
• 6/10 showed an increase of FL-SMN between
  40-300
• Therapy with VPA in SMA patients

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Individual therapy of SMA patients with VPA

• Individual therapies of about 40 type I-III SMA
  patients (seen by various neuropediatricians,
  neurologists, pediatricians in Germany and
  Austria)
• Patients have a level of VPA in serum of
  70-80µg/ml
• Most patients were also substituted with
  L-carnitin (50-100 mg/kg/day)
• The younger the patients are the more
  improvements were reported
• First improvements usually after 6 months of
  therapy
• There were no significant negative side effects
  reported so far
• Results of quantitative measurements of FL-SMN
  showed an up-regulation in about 50 of patients

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Future trials with VPA

• Germany type I SMA patients
• Extinction on European level
• USA type II and III SMA patients
• Starts in June 2005
• EU type II SMA patients
• Planed, next meeting of the ENMC in autumn

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Cologne Lars Brichta Eric Hahnen Markus
Riessland Irmgard Hölker Bonn Yvonne Hofmann
Karsten Haug Yuli Sun Heidrun Raschke Thomas
Klockgether Sebastian Stier
Erlangen Florian Siebzehnrubl Ingmar
Blümcke Ilker Eyupoglu Hannover Kirsten
Haastert Peter Claus
ZMMK
Köln Fortune